The present invention is in the field of molecular biology. More particularly, the present invention relates to a novel lysolipid receptor, a human EDG-4 receptor, a method of identifying lysolipid receptors involved in inflammatory response and the lysolipid receptors so identified, and a method of identifying ligands which interact with such lysolipid receptors.
(a) EDG Receptors
EDG receptors have been grouped with orphan receptors because their endogenous ligands are not known (for example see Hla T and Maciag T (1990) J Biol. Chem. 265:93018-13; U.S. Pat. No. 5,585,476). Recently, however, lysophosphatidic acid (LPA) has been demonstrated to be the endogenous ligand for the EDG-2 receptor (Hecht et al. (1996) J. Cell. Biol. 135: 1071-1083; An et al. (1997) Biochem. Biophys. Res. Comm. 213: 619-622).
The EDG receptors are seven transmembrane G protein coupled receptors (T7Gs or GPCRs). GPCRs are so named because of their seven hydrophobic domains of 20-30 amino acids which span the plasma membrane and form a bundle of antiparallel xcex1 helices. These transmembrane segments (TMS) are designated by roman numerals I-VII and account for structural and functional features of the receptor. In most cases, the bundle of helices forms a binding pocket; however, when the binding site must accommodate more bulky molecules, the extracellular N-terminal segment or one or more of the three extracellular loops participate in binding and in subsequent induction of conformational change in intracellular portions of the receptor. The activated receptor, in turn, interacts with an intracellular G-protein complex which mediates further intracellular signaling activities such as the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, activation of protein kinases, alteration in the expression of specific genes.
When the receptor is activated by the binding of a ligand, the conformation of the receptor changes allowing it to interact with and activate a G protein. The activated G protein causes a molecule of guanosine diphoshate (GDP), that is bound to the surface of the G protein, to be replaced with a molecule of guanosine triphosphate (GTP), which causes another alteration in the conformation of the G protein. With GTP bound to its surface the G protein can regulate the activity of an effector. These effectors include enzymes such as adenylyl cyclase and phospholipase C and certain transport protein and ion channels such as calcium ions, potassium ions or sodium ions.
GPCRs are expressed and activated during numerous developmental and disease processes. Identification of a novel GPCR provides the opportunity to diagnose or intervene in such processes. The receptor can be used in screening assays to identify physiological or pharmaceutical molecules which trigger, prolong or inhibit a receptor""s activity or a differentially modulate distinct intracellular pathways which are controlled by GPCRs. However, for many of the GPCRs (such as the EDG receptors) the biological processes mediated by the receptor are currently unknown. There exists a need therefore for methods to identify the biological processes mediated by these GPCRs and also for methods of identifying other GPCRs that may be involved in these processes.
Because there are diverse functions of GPCRs, it is not surprising that there are a number of therapeutic drugs that act by modifying the function of GPCRs. Therapeutic drugs which modify the GPCRs are particularly attractive because of the ability to design such drugs with particular specificity so that they turn on or off specific receptors and their signaling pathways.
(b) Lysophogpholinids and Inflammation
LPA is a naturally-occurring agonist of the EDG-2 receptor (Hecht et al. J Cell Biol 135: 1071, 1996). LPA, and many other lysophospholipids, are produced by activated platelets as a consequence of inflammation-related intracellular signal transduction accompanying aggregation and thrombus formation. Similar inflammatory pathways occur in many cell types, and typically lead to production of LL and other lipid mediators within seconds to minutes, and activation of new gene expression within minutes to hours.
A number of lysop pholipids have been studied to determine their biological effects. For example, the lysophospholipd sphingosine-1-phosphate (S1P) appears to play a role in a number of CNS-related biological processes. These include apoptosis, mitogenesis and cytoskeletal reorganization. S1P has been proposed to mediate at least some of the biological functions of PDGF and NGF. The former is a growth hormone with potent mitogenic and wound-healing activity. The latter is a neurotrophic factor, which has also been proposed to play a role in neuropathic pain.
In addition, it has been reported that there is activation of NF-xcexaB by S1P in U937 cells; however, the authors assumed that S1P was an intracellular second messenger, and no attempt was made to determine whether this response was receptor-mediated. Furthermore, the functional relevance of NF-xcexaB activation was not tested, e.g. by examining the possible upregulation of inflammatory cytokines, adhesion molecules or other NF-xcexaB-dependent genes. If multiple receptors for S1P exist, the finding of NF-xcexaB activation offers no utility by itself, since one, several, or all of the receptors might respond through NF-xcexaB.
Moreover, direct modulation of NF-xcexaB activation cascades has been proposed as a therapeutic mechanism for inflammation or apoptosis. However, NF-xcexaB plays a vital role in innate immunity against ubiquitous microbial pathogens and in mobilizing the antigen-specific immune system. Therefore, rather than targeting this irreplaceable defense system, it would be preferred to instead block inappropriate activation of NF-xcexaB by specific inflammatory or apoptotic signaling events. Accordingly, it is highly desireable to design therapeutic agents which could modulate NF-xcexaB activation and thereby prevent unwanted apoptosis or thereby enhance immune function in immunocompromised hosts via a receptor modulated pathway.
It has now been discovered that there are LL/EDG receptors which are involved in an inflammatory response signaling pathway and an apoptotic signaling pathway. In particular, it has been discovered that the EDG-2, EDG-3, EDG-4, EDG-5 and EDG-6 receptors activate NF-xcexaB and/or the production of IL-8. Accordingly, the present invention provides a link between NF-xcexaB activation and edg receptors and hence a means for controlling NF-xcexaB activation and thereby for controlling apoptosis and inflammatory responses.
In an aspect of the present invention, it has been discovered that agonists to the EDG-2, EDG-5 and EDG-6 receptors result in activation/production of NF-xcexaB and/or IL-8. In particular, it has been discovered that LPA will act as an agonist to the EDG-2, EDG-5 and EDG-6 receptors resulting in activation/production of NF-xcexaB and/or IL-8.
In another aspect of the present invention, it has been discovered that agonists to the EDG-3 and EDG-4 receptors result in activation/production of NF-xcexaB and/or IL-8. In particular, it has been discovered that S1P and SPC will act as an agonist to the EDG-3 and EDG-4 receptor resulting in activation/production of NF-xcexaB and/or IL-8.
In another aspect of the present invention there is provided isolated polynucleotides encoding the human EDG-4 receptor. The isolated polynucleotides may be either cDNA or genomic clones.
In particular, the present invention provides an isolated nucleotide sequence selected from the group consisting of:
a) the nucleotide sequence comprising nucleotides 38-1099 of FIG. 15A (SEQ ID NO:1);
(b) the nucleotide sequence of FIG. 15B (SEQ ID NO:2);
(c) a nucleotide sequence with at least about 95% sequence identity to (a) or (b) and which hybridizes under stringent conditions to sequences (a) and (b), respectively;
(d) a nucleotide sequence which encodes the amino acid sequence of FIG. 16A (SEQ ID NO:3) for the human EDG-4 receptor; and
(e) a nucleotide sequence which encodes the amino acid sequence of FIG. 16B (SEQ ID NO:4) for the human EDG-4 receptor.
There is also provided: expression vectors; host cells; purified amino acid sequences; complementary nucleic acid sequences; biologically active fragments; and hybridization probes, for such nucleotide sequences and their encoded amino acid sequences.
In another aspect of the present invention, there is provided a method of determining whether a DNA sequence encodes edg receptors that are involved in inflammatory response by measuring the induction of NF-xcexaB and/or IL-8 upon activation by a suitable ligand.
In another aspect of the present invention, there is provided a method of determining whether a DNA sequence encodes an edlfosine receptor that is involved in inflammatory response by measuring the induction of NF-xcexaB and/or IL-8 activation by a suitable ligand, including edelfosine.
In another aspect of the present invention, there is provided a method of identifying ligands that interact with edg or lysolipid receptors that are involved in inflammatory response. In particular, the present invention provides a method of identifying ligands which interact with edg or lysolipid receptors by measuring the induction or lack of induction of NF-xcexaB and/or IL-8.
In another aspect of the present invention, there is provided a method of modulating or treating an inflammatory process condition in a subject by administering an effective amount of a pharmaceutical composition comprising an agonist or antagonist of an NF-xcexaB and/or IL-8 modulated EDG or lysolipid receptor and a pharmaceutically acceptable excipient, for upregulation or downregulation of the inflammatory process, respectively. In particular, agonists and antagonists of the EDG-2, EDG-3, EDG-4, EDG-5 and/or EDG-6 receptor are applicable.
In another aspect of the present invention, there is provided a method of modulating an immune response in a subject by administering an effective amount of a pharmaceutical composition comprising an agonist or antagonist of an NF-xcexaB and/or IL-8 modulated EDG or lysolipid receptor and a pharmaceutically acceptable excipient, for upregulation or downregulation of the immune response, respectively. In particular, agonists and antagonists of the EDG-2, EDG-3, EDG-4, EDG-5 and/or EDG-6 receptor are applicable.
In another aspect of the present invention, there is provided a method of controlling apoptosis by activating an EDG or lysolipid receptor which receptor activates the induction of NF-xcexaB. In particular, by modulating the EDG-2, EDG-3, EDG-4, EDG-5 and/or EDG-6 receptor via agonists or antagonists there is provided a method of controlling apoptosis.
An EDG receptor herein refers to any receptor with at least 27-30% identity, preferably at least 30-35% identity, more preferably at least 35-40% identity, even more preferably at least 40-45% and most preferably at least 45-50% identity with each other. As is known in the art, the percentage identity of the amino acid sequences of related receptors is generally greater in the same species than in different species.